Food compositions



United States Patent FOOD COMPOSITIONS Paul Gyorgy, Villanova, Pa., and Richard Kuhn and Friedrich Zilliken, Heidelberg, Germany, assignors, by mesne assignments, to American Home Products Corporation, New York, N. Y., a corporation of Delaware No Drawing. Application April 22, 1952, Serial No. 283,773

4 Claims. (Cl. 99-54) This invention relates to food products and, more particularly, to food products which are suitable for use in the feeding of infants.

Our invention is more especially concerned with certain new and improved food compositions which contain the usual fat, protein, carbohydrate, fatty acid, amino acid, vitamin and mineral constituents necessary for growth of the infant, but which compositions also contain, in addition, an added substance supplying a specific factor or principle necessary to support the growth of the microorganism Lactobacz'llus bifidus as the predominant microorganism in the digestive tract of the infant fed therewith.

There are a number of commercially available food compositions utilized for the feeding of infants which are based on cows milk as an essential and important nutritive constituent thereof. These compositions generally contain, in addition to added vitamins, a fat constituent, a milk protein, an edible sugar (frequently lactose), minerals, and ash. The relative proportion of these constituents is such as to provide a nutritive formula meeting all the nutritional requirements of the infant when an aqueous solution of a specified concentration is prepared and fed to the infant.

As an example, one well-known commercially available infant feeding formula, derived from cows milk but having major physical and chemical properties essentially the same as human milk, contains approximately 1.5% of protein, 7.0% of carbohydrate, 3.5% of fat, 0.38% of ash and a mineral constituent, such as iron, present in a relatively minor amount as, for example, approximately 0.0005%, as the food is made up as a liquid ready to feed to the infant. Such a composition is, in many respects, similar to human milk, will supply approximately 20 calories per fluid ounce, and contains of course, in addition, various vitamin constituents, such as vitamin A, thiamin, riboflavin, vitamin C, vitamin D and niacin, all present (with the exception of vitamin C) in amounts considerably in excess of the amounts in which those vitamins are present in an equal volume of human or breast milk.

It should be understood, of course, that the particular commercial food composition having the foregoing analysis is but one of many available infant feeding formulas, all based on, or derived from, cows milk, which compositions are usually supplied, either in a concentrated liquid form which is adapted to be diluted with water before use, or as a solid powder or granular composition which is dissolved in water in specified proportions to provide solutions of specified concentrations. Such compositions will vary to some extent from each other in the ingredients utilized, i. e., the source of the fat present therein, the content of saturated and unsaturated fatty acids and amino acids, the carbohydrate constituents, the relative proportions of the ingredients, etc., but they are essentially similar in that all are derived from the milk of cows.

While some of these commercially available infant food compositions are similar to human milk, to greater or less degree, in their major physical and chemical properties, there are, however, certain important differences in the predominating microorganisms present in the intestinal bacterial flora characteristic of an infant fed exclusively with human or breast milk, as compared with the intestinal flora of an infant who is fed with one of the commercial food formulas based on, or derived from, cows milk. The intestinal flora of the breast fed infant "ice is characterized by the presence therein of the microorganism Lactobacillus bifidus as the predominating microorganism. The stools of infants who have been fed exclusively with human milk are in fact remarkable in that this microorganism comprises practically the entire bacterial flora present in the excretions.

As seen in smears when freshly isolated from the infant feces the L. bifidus organism appears as grampositive, straight or curved rods that are non-motile and that do not form spores. One or both ends may appear to be split longitudinally to give the eflect of two short branches, this appearance leading to the term bifid. One end of the organism may be bulbous or racketshaped.

While this microorganism predominates in the intestinal tract and feces of infants fed with human milk from lactating mothers, it is not the characteristic or predominating microorganism in the intestinal tract of infants fed with other foods, such as cows milk, or with the commercially available food preparations derived from, or based on, cows milk. Infants so fed show a less uniform intestinal flora with an appreciable proportion of gram-negative rods and cocci.

The fact that a stable L. bifia'us flora is characteristic only of the intestinal tract of breast-fed infants is now fully understood and appreciated by pediatricians and other specialists in the medical, biological, and nutritional sciences. See, for example, the publications of Tissier, Comptes rendus soc. biol., 51, pages 943-945; and 60, pages 359-361; and Ann. inst. Pasteur, 19, pages 109-123; and 22, pages 189-207. See also the papers of Moro; Wien, klin, Wochschr.; 13, pages 114-115, and Jahrb. Kinderheilk, 61, pages 687-734; and 61, pages 870-899. That the original observations of Tissier concerning the predominance of L. bifidus in nurslings stool have been amply confirmed is pointed out in the more recent publication of Norris, Flanders, Tomarelli and Gyorgy in the Journal of Bacteriology, 60, pages 681-696 (December 1950).

Medical authorities are now in general agreement that many of the advantages which a breast-fed infant seems to have, insofar as its nutrition and resistance to certain diseases are concerned, are attributable, at least in part, to the predominance of L. bifidus in his intestinal tract. The prevalence of this organism in the intestinal tract is now believed to be of physiological significance, and its growth may prevent intestinal disorders caused by other bacteria. Resistance to bacterial intestinal diseases, and even to parenteral disorders, such as respiratory infections, is typical of breast-fed infants. Infants who are bottle-fed with the milk of cows and other animals, or with infant feeding formulas utilizing cows milk as the source of protein, are definitely more susceptible to such disorders. In this connection see the article of Robinson in Lancet, entitled: Infant Morand Mortality, vol. CCLX, page 788 (April 7,

In the infant colon in which a stable L. bifidus flora is lacking it is now generally believed that the infant is more likely to suffer from disorders attributable to the growth of other bacteria, some of which are harmful to the infant, since competition for the available food supply in the infant intestinal tract where L. bifidus flourishes reduces the possibility that other bacteria, particularly the harmful B. coli, may grow in considerable numbers or in extensive colonies. In any event, where the gram-positive bacillus L. bifidus is present in large numbers in the infant intestine, which is now found only when the infant is breast-fed, there is considerably less growth of other microorganisms, particularly of the gram-negative B. 00111, the Clostridia, and certain airborne microorganisms such as the Sarcinae bacteria. While the reasons for the superior resistance to disease of breast-fed infants as compared with infants who are fed on cows milk, or on the usual infant formulas in which cows milk is a principal constituent, are not fully understood, pediatricians are generally agreed that in some measure, at least, the superior resistance is attributable to the presence of an extensive L. bifidus flora in the intestines of nurslings, this bacterium being absent in any considerable numbers from the intestines of infants who are not fed with human milk. Interest in the isolation and determination of the growth requirements of L. bifidus has therefore been stimulated by its apparently unique predominance in the intestinal tract of the breast-fed infant.

Extensive research work has been carried out on the identification and isolation of the active biological principle which is responsible for promoting the growth of L. bifidus in the intestines of infants-who are nourished with human milk, which active principle is evidently not present in sufficient amount to insure :the growth of L. bifidus as the predominant microorganism in the intestinal tract of infants who are fed with cows milk, or with any of the usual feeding compositions or infant formulas based on cows milk. Our copending application Serial No. 283,006, filed April 18, 1952, is directed to a process for recovering specific growth-promoting substances for L. bifidus from human milk, and to the novel growthpromoting substances thereby recovered. This research has demonstrated that the specific growth-promoting substances or factor is probably also present, at least to some extent, in cows milk as well as in human milk, but the amount contained in cows milk is so small as to be only a very small percentage of the amount contained in an equal volume of human milk. It is far insufficient in quantity to provide the infant fed therewith with enough of the growth-promoting substances or factor to insure dominance of the L. bifidus micro organism in his colon andfeces.

While the concentration of the .specific growth promoting factor for L. bifidus in coWs milk varies to some extent with various samples of milk, and to some extent with the particular strain or variety of the L. bifidus microorganism which is selected for the test, ordinarily the specific growth-promoting factor is present in cows milk only in about A the concentration (approximately 3.33%) in which it is present in human or breast milk. This concentration is evidently too low to insure that L. bifidus will be the predominating microorganism in the intestinal flora of infants fed with cows milk. As only about one-half of the solid content of the commercial infant food preparation of which the analysis is given above is cows milk, the L. bifidus growth promoting factor is actually present in such an infant food formula only to the extent of approximately ,6, (approximately 1.67%) of the amount in which it is present in an equal volume of human milk.

While the advantages of supplying the feeding infant with a food composition suitable for promoting the growth of L. bifidus as the predominating microorganism in the infant colon have been appreciated for some time, it has not been possible to attain this with any of the presently available food compositions wherein the specific growth-promoting factor, on the average, has been present only to the extent of approximately 1.67% of the amount in human milk as compared on an equal volume basis. From time to time thought has therefore been given to the possibility of adding to the infant food com positions some material or substance which would supply the specific L. bifidus growth-promoting substances or factor in greater amount, so that this growth-promoting factor could be present in the infants nourishment to an extent sufficient to insure the predominance of the microorganism L. bifidus in his intestinal tract. Previously, however, no substance other than human milk has been known to contain the specific growth-promoting substances or factor in sufiiciently high concentration to permit increasing the amount of this biologically-active principle to an extent sufiicient to insure this result with those preparations currently available, and human milk, of course, is not a practical additive to such formulas.

We have now discovered that the specific growthpromoting factor necessary to support the growth of Lactobacillzrs bifidus in the infant intestinal tract, or at least necessary to support the growth of these strains of the microorganism which have hitherto been known to grow only in the presence of human milk (or the specific growth-promoting substances contained therein), can be supplied by including in the infant food composition a small amount of hydrolyzed chitin. Specifically, we prefer to include in the infant food composition hydrolyzed chitin as prepared from lobster shell, or from crabs, insects, mushrooms, etc., the chitin being first recovered from the lobster shell or other source by a suitable treatment, and this chitin then subjected to hydrolysis, preferably by treatment with a strong mineral acid, to yield the hydrolyzed product found to possess Luctobacillus bifidus growth-promoting activity.

For example, lobster shell may be first treated, preferably for one or two days, with a caustic solution such as a ten percent solution of sodium hydroxide. After the caustic treatment the lobster shell is subjected to the action of a mineral acid, such as hydrochloric acid of about 5% concentration, for a period of one or two days, at the end of which a skeletonized product is secured which is practically pure chitin.

The chitin is first hydrolyzed, preferably at room temperature, by treatment with a strong mineral acid, such as sulfuric acid of approximately 50% concentration. After dilution with water, as for example with six parts of water for each part of hydrolyzed chitin, the product is de-ionized, preferably by treatment with ion exchange resins, or otherwise, and the inactive material is precipitated, as by the addition of ethanol until the ethyl alcohol concentration is up to or by the addition of ether until no more precipitate isformed. After precipitation and removal by filtration of the almost inactive material, the material which is effective in promoting the growth of L. bifidus is in the filtrate. This filtrate may be evaporated to dryness, or otherwise treated to obtain the active material in solid form, or, if desired, the product need not be recovered in solid form but may be added to the infant food composition in the form of a solution, preferably a concentrated solution.

As a specific example of the preparation of a material possessing growth-promoting activity for L. bifidus from lobster shell, or from chitin as obtained from lobster shell, the following may be regarded as illustrative of a preferred procedure.

700 grams of chitin as obtained from lobster shell may be treated with a cooled mixture of 500 milliliters of water and 500 milliliters of concentrated sulfuric acid. It is allowed to stand at about room temperature (20 C.) for about 43 hours, during which time the solution is stirred or agitated occasionally.

Water in the amount of five liters is then added to the mixture, with cooling, and the solution then decolorized by treatment with a small amount of decolorizing carbon (Norit). The sulfuric acid is next removed from the clear, colorless solution by treatment with an ion exchange resin (for example, by treatment with IR4B, sold by Rohm and Haas Co.), or by precipitation of the sulfate ion as barium sulfate. The solution, after filtration or centrifugation, may then be evaporated to dryness, preferably at a reduced pressure, less than atmospheric.

The solid residue remaining is then dissolved in 60 milliliters of water, and 1020 milliliters of absolute ethanol are then added to the solution. A precipitate appears, and is removed. To the filtrate remaining, dry ether is added, the addition being continued until no more solid material separates. When the alcohol-ether solution is subjected to evaporation, an additional crystalline precipitate showing only a small amount of growthpromoting activity is secured.

After crystal deposition ceases on continued evaporation, the crystals are removed and the filtrate is evaporated to dryness at a reduced pressure, less than atmospheric. An amorphous residue .is obtained which is highly active. Upon boiling this residue with enough methanol to dissolve it, followed by cooling the solution at about 0 C. for several days, a crystalline product in the form of fine plates separates. This is highly active in promoting the growth of L. bifidus. About 80-100 micrograms of the material possess a growth-promoting activity which is equivalent to one E unit, where one B unit is the growth response given by approximately 0.06milliliters of human milk of average content of the growth-promoting factor.

The nitrogen content of the hydrolyzed chitin product, as recovered, is 5.0%. Its melting point is 130133 C. In water solution at a concentration of 0.05%, the [a] value is Hydrolyzed chitin is extremely active as a source of the specific growth promoting factor, having an activity approximately forty times that of the most active simple chemical compound known to possess said activity, 2- desoxy-Z-acetamino-d-glucose, when compared on an equal weight basis. As a matter of fact'hydrolyzedchitin, in addition to being a crystalline substance, thereby rendering it very desirable as an additive to food preparations, is also much more active than human milk when compared on an equal weight basis.

As illustrative of the relative order of activities, from approximately 0.08 to 0.10 milligram of hydrolyzed chitin as recovered from lobster shell shows an activity of 1 unit, this being usually expressed as a mg./E ratio of 0.08 to 0.10. The standard of activity, E, is the activity for promoting the growth of Lactobacillus bifidus exhibited by approximately 0.06 milliliter of human milk of average or usual activity. (The growth promoting activity of breast milk varies to some extent, and the value, E, is based on average conditions.) The lower the mg./E value for a given material, the higher is its L. bifidus growth promoting activity.

We have observed that the mg./E value for N-acetylglucosamine (2-desoxy-2-acetamino-d-glucose) is approximately 2.03.0. Its activity in promoting the growth of L. bifidus is therefore only about one-fortieth that of hydrolyzed chitin as recovered from lobster shells. Human milk is also considerably less active, certain concentrated samples of human milk, for example, when tested, having mg./E values of from 2.3 to 3.0. Blood group substances A and B, showing a mg./E of approximately 0.60, are also far less effective on an equal weight basis for promoting the growth of Lactobacillus bifidus than is hydrolyzed chitin from lobster shell.

It is indeed remarkable that a white crystalline material, in form very desirable as an additive to food preparations such as infant feeding preparations, possesses such high activity for promoting the growth of L. bifidus, especially those strains of the microorganism which grow only in media which contains such growth promoting substances.

We have found that the amount of hydrolyzed chitin derived from lobster shell that should be incorporated in an infant feeding formula or composition based on or derived from cows milk of the general type previously described will ordinarily range from about 0.1% to 5.0% by weight, this percentage being based on the total weight of solids present in the infant feeding formula or composition. Frequently an amount of added hydrolyzed chitin not exceeding about 1.0% by weight will be found satisfactory for many purposes in infant feeding.

The addition of hydrolyzed chitin to the infant feeding formula in amounts within the range 0.1% to 5% by weight thereof, based on the solids content basis, supplements the small amount of specific L. bifidus growthpromoting factor already present in that composition as a result of its cows milk content so that the quantity of growth-promoting principle, as supplemented, is approximately comparable with the amount occurring on the average in an equal volume of human or breast milk. When the infant is fed with this composition or formula, the protein content thereof being derived from cows milk, this food being supplemented, however, as to its specific growth-promoting factor by the addition thereto of hydrolyzed chitin, all strains or variants of the L. bifidus microorganism present in the infants intestinal tract have available for their growth in the infant colon a source of nutriment which, as respects the amount of growth-promoting substances, is substantially the same as human milk. The L. bifidus microorganism is thus enabled to grow and proliferate in the intestinal tract of the infant to the same extent and degree that it would were the infant nourished entirely upon human milk.

Various changes and modifications may be made in our invention as described, and to the extent that these changes and modifications are within the scope of the appended claims, they are to be regarded as part of our invention.

We claim:

1. A foodstuff comprising a nutritive composition which is normally deficient in Lactobacillus bifia'us growthpromoting substances, said foodstuff containing, as a supplemental source of said growth-promoting substances, hydrolyzed chitin.

2. A foodstuff comprising a nutritive composition which is normally deficient in Lactobacillus bifidus growthpromoting substances, said foodstuff containing, as a supplemental source of said growth-promoting substances, from 0.1% to 5.0% of hydrolyzed chitin, said percentages being by weight, based on the solids content of said foodstuff.

3. An infant foodstuff which comprises proteins, carbohydrates and fats, said foodstuff also containing, as a supplementary source of growth-promoting substances for Lactobacillus bifidus in the intestinal tract of infants fed therewith, hydrolyzed chitin.

4. An infant feeding composition including proteins, carbohydrates and fats, said feeding composition containing from 0.1% to 5.0% of hydrolyzed chitin as a source of growth-promoting substances for Lactobacillus bifidus when said composition is fed to infants having said microorganism present in their intestinal tracts, said percentages being by weight, based on the total weight of solids in said composition.

References Cited in the file of this patent UNITED STATES PATENTS Name Date Nielson Nov. 23, 1926 Block et al. Apr. 24, 1945 OTHER REFERENCES Number 

1. A FOODSTUFF COMPRISING A NUTRITIVE COMPOSITION WHICH IS NORMALLY DEFICIENT IN LACTOBACILLUS BIFIDUS GROWTHPROMOTING SUBSTANCES, SAID FOODSTUFF CONTAINING, AS A SUPPLEMENTAL SOURCE OF SAID GROWTH-PROMOTING SUBSTANCES, HYDROLYZED CHITIN. 